Environment Industrial

Studies on mutagenesis in vitro by agents that generate oxygen free radicals have used both reversion assays (Loeb, et al., 1988; Cheng, et al., 1991; and for ward mutation assays (McBride et al., 1991).  Reversion assays measure the frequency and types of mutations at a defined mutant locus on a DNA molecule.  Forward mutation assays are inherently less sensitive, but provide a wide spectrum of mutations and sequence contexts produced as a result of DNA damage by oxygen free radicals.

So far, our analysis indicates that many agents that generate oxygen free radicals cause mutations in vitro.  This list includes transition metals, myeloperoxidase and stimulated polymorph nuclear leukocytes (Reid and Loeb, unpublished).  Enhancement in mutation frequencies have been observed with (Tkeshelashvili, et al., 1991).  It should be noted that there is evidence for mutagen city in vivo with iron, copper, and cobalt as well as manganese (Sir over and Loeb, 1976).  Nickel and chromium are classified as human carcinogens (Sunderman, 1986) and evidence has been presented association increased iron stores in humans with the risk of developing certain tumors (Stevens et al., 1986).

It may be instructive to analyze the mutagenic spectrum produced by exposure of DNA to copper in vitro.  Using a forward mutation assay (Kunkel, 1984), one can detect a wide variety of nucleotide sequence alterations produced within a portion of the lacZgene and its regulatory region.  Mutations in the gene are seen as light blue or white plaques after transfect ion of E. coli and plating on an indicator dye.  The nature of the mutations is determined by DNA sequencing.  The spectrum of mutations produced by exposure of SNA to copper is given (Tkeshelashvili, et al., 1991).  Of the mutations induced by Cu+ and Cu2+, 90% are single-based substitutions.  Mutations are not random.  There are hot spots at positions+107, +108, +129 and +145.  Nucleotide sequence alterations at positions of tandem single-based substitutions have been reported so far only in the case of UV mutagenesis (Miller, 1985; Schaaper, et al., 1987) and these could represent a diagnostic marker for mutagenesis by agents yielding oxygen free radicals.

By compiling the mutagenic spectrum of agents that induce oxygen free radicals using single-stranded DNA, one can make the following generalizations:  The most frequent substitutions are C→T, presumably caused by damage to C so that it base-pairs with A at high frequency.  Chemical structure of the C modification that results in C→T transitions remains to be established.  The second most frequent types of substitutions observed with single-stranded DNA are G→T Tran versions.  These could be mediated by the binding of metals such as copper irons to the N7-position of guanine in DNA, resulting in depurination or through the production of 8-hydroxyguanosine (Shibutani, et al., 1991; Wood, et al., 1990; Cheng, et al., in press).  Thirdly, there are hot spots throughout the DNA that could result from localized metal binding in a specific sequence context and the generation of oxygen free radicals at that site.  Alternatively, specific hot spots could be the site of enhanced sensitivity to damage by reactive oxygen species.  Lastly, tandem C→T substitutions are unusual.  If similar mutations are documented with other agents that generate oxygen free radicals, they could serve as a diagnostic marker for oxygen mutagenesis.

Based predominately on epidemiological studies, only a small number of chemicals have been identified definitively as human carcinogens. For the most part, individuals exposed to high concentrations of these particular chemicals have exhibited an unusually high incidence of a specific tumor. There is increasing evidence that human cancers are causally associated with mutations in somatic cells. In vitro studies have shown that many of these chemicals form covalent adducts on DNA that alter DNA’s base-pairing properties. If these DNA modifications are not repaired, the altered DNA is copied at the time of cell replication, leading to misincorporations that produce mutagenesis (Loeb, 1989).  Included in the catalogue of human carcinogens are a number of metals that are ubiquitously present in our environment (Sunderman, 1986).  These metals form weak interactions with DNA and there is evidence that damage to DNA by transition metals is mediated by the generation of oxygen free radicals.  In this manuscript we will first summarize current concepts on the etiology of cancer by environmental agents and then consider in some detail the types of mutations caused by metal-generated oxygen free radicals.

In considering the spectrum of environmental agents that are classified as human carcinogens it is obvious that tobacco smoke is the most significant.  In the United States, cigarette smoking is responsible for some 30% to 40 of cancer deaths (Loeb et al., 1984) Cancer of the lung as a result of cigarette smoking is epidemic.  Until recently, the incidence of death from cancer of the lung in U.S. males had risen progressively for over forty years.  In the last ten years, the prevalence of cigarette smoking among males in the United States has declined from a high of 50% in 1965 to the current level of less than 30%.  As a result, after forty years of relentless increase in male lung cancer deaths, the rate has plateaued; in the 45-55 year old range there has been a significant decrease Figure 1. Unfortunately, the prevalence of smoking has not decreased among American females and currently cancer of the lung outranks cancer of the breast as the leading cancer-related cause of death in women in the United States.  In emerging industrialized countries, such as Thailand, the incidence of tobacco smoking is rapidly increasing.  If the experience in the United States is of any prognostic value, the lung cancer rate in Thailand will rapidly accelerate and lung cancer will soon become one of the leading causes of death.  Hopefully, Thailand can use the experience of the United States to curtail this epidemic.

Mutations and Cancer

A current model linking DNA damage to mutagenesis and carcinogenesis is presented with Figure 2.  In this model, random mutations result from DNA polymerase errors opposite unrepaired DNA damage.  Mutations found in key genes program changes in cells and initiate the carcinogenic process. These genes include oncogenes as well as genes involved in the maintenance of DNA replication fidelity (Loeb, L.A., 1989), and chromosomal segregation (Hartwell and Weinert, 1989).  DNA damage occurring in cells that do not divide, such as neurons, cannot be propagated to successive generations and thus is not associated with malignancy.  Also mutations do not result from an error-free DNA repair or from unrepaired DNA damage tat is copied during cell replication without a change in sequence.  A major factor linking DNA damage to mutagenesis is the copying of damaged DNA by DNA polymerases during each cell division cycle; DNA polymerases copy past the damaged DNA and insert non-complementary nucleotides opposite the site of damage.  In prokaryotes, SPS error prone DNA repair synthesis (Walker, 1984) can be induced by DNA damage, possibly to facilitate replication past DNA lesions, but it is uncertain whether such a response exists in human cells.  A few of the randomly distributed mutations would be in key genes that alter the growth properties of cells, allowing them to escape homeostatic mechanisms that regulate cell division and mutations in genes that might promote metastasis.  In this scheme, any process that increases the likelihood of division in cells containing DNA damage would also increase the likelihood of mutations and malignancy.  Stimuli for cell division could include increases in autocrine and paracrine growth factors, viruses, and regenerative stimuli brought about simply by the death of adjacent cells and tissues.  This model links unrepaired DNA damage to increased cell replication as causative factors in human cancer.